Figure 2.

Equilibrium binding constant (K_d) of K-loop mutants. Mean and SD of at least three independent experiments are shown. (A) K_d of KK1 (green), CK6 (red), CK1 (blue), and KK6 (black) in various nucleotide conditions. Binding energy ΔG = k_BT log K_d is shown on the right axis. (B) K_d of K-loop mutants in the s- and the w-states plotted against N, the number of positively charged residues in the K-loop. Black circle, conventional kinesin mutants in ADP state; blue square, conventional kinesin mutants in AMP-PNP state; green circle, KIF1A mutants in ADP state, and red square, KIF1A mutants in AMP-PNP state. (C) ΔΔG, difference of the binding energy between the s- and the w-states plotted against N. Both conventional kinesin (blue square) and KIF1A (red circle) mutants were on the same line with a slope −0.25 k_BT. (D) K_d of CK6 to subtilisin-digested MT plotted against the concentration of subtilisin (molar ratio to tubulin). K_d^w of CK6 (red) increased according to the degree of the subtilisin digestion to the level similar to K_d^w of CK1 to intact or digested MT (green), whereas K_d^s of CK6 (blue) was not significantly affected by the subtilisin digestion. (Lower) The degree of the tubulin digestion by subtilisin. The anti-α tubulin antibody recognized an epitope upstream to the subtilisin digestion site, thus subtilisin-digested α tubulin is detected as a band with higher mobility (lower band in α). The epitope of the anti-β tubulin antibody was in the subtilisin-digested region, thus subtilisin digestion results in the loss of the band.